This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Subproject #3: Determine the mechanism by which the EHV-1 IR2 protein inhibits viral gene expression and replication Seong K. Kim Equine herpesvirus 1 (EHV-1) is an important pathogen of equine and a useful model to investigate Alphaherpesvirus gene regulation as its gene program is initiated by expression of a single immediate-early (IE) gene that activates expression of 50 early (E) genes which include E regulatory genes IR2, UL5, EICP0, and IR4. The unique EHV-1 IR2 protein (IR2P) is a 1,165-amino acid truncated form of the immediate-early protein (IEP) and lacks IEP residues 1 to 322 that harbor the trans-activation domain (TAD) essential for trans-activation and viral growth. IEP is multi-functional, and our libraries of IE mutants and 17 IE mutant viruses allowed us to identify and characterize several functional domains essential for EHV-1 replication: trans-activation domain (TAD), the serine-rich tract (SRT), DNA-binding domain (DBD), nuclear localization sequence (NLS), and domains that interact with other EHV-1 proteins or cell proteins, including transcription factors TBP and TFIIB. Transient transfection assays showed that the early regulatory IR2P by itself down-regulated the IE promoter and all early promoters tested and abrogated activation of viral promoters mediated by the IEP and the early regulatory protein UL5P in a dose-dependent manner. The IR2P physically interacted with the general transcription factors TFIIB and TBP. Virus growth assays revealed that the IR2P inhibited virus production by up to 90-fold in equine NBL6 cells. On the basis of these findings, we hypothesize that IR2P functions as a dominant-negative regulator of EHV-1 gene expression by blocking IEP-binding to viral promoter sequences and/or squelching the limited supplies of TFIIB and TBP. Our overall question is how does IR2P inhibit viral gene expression and replication? In this proposal, we are characterizing the biological and molecular properties of IR2P in order to define the mechanism(s) by which IR2P inhibits EHV-1 gene expression and replication.